Journal of Clinical Laboratory Analysis 1 :77-79 (1987)

Homogeneous Enzyme lmmunoassay for Macromolecular Antigens Using Hybrid

Anti body Yoshihiro Ashihara, lsao Nishizono, Hiromasa Suzuki,

Research Laboratories, Fujirebio, Inc., Komiya-cho Hachioji, Tokyo 192, Japan

and Yasushi Kasahara

A new homogeneous enzyme immunoassay for the determination of macromolecular anti- gens has been developed based upon com- petitive enzyme inhibition using hybrid antibody- containing anti-ligand and anti-enzyme inhibitory antibodies.

The hybrid antibody was prepared by the reaction of anti-glucose-6-phosphate dehydro- genase (G6PDH) mouse IgG maleimide with anti-human IgM goat IgG-SH. In the absence of the antigen, the hybrid antibody inhibits the

enzyme activity. On the other hand, in the pres- ence of an excess amount of antigen, the hybrid antibody binds to the antigen and is unable to bind with the enzyme.

The hybrid antibody inhibited 80% of the original activity of enzyme and saturated the inhibition of G6PDH within 10 min. The enzyme activity was proportional to the concentration of human IgM. The measurable range for IgM was 78 pgiml to 1 mg/ml. Human IgG present in the serum did not affect this method.

Key words: Hybrid antibody, homogeneous enzyme immunoassay, macromolecular antigens, glucose-6-phosphate dehydrogenase

INTRODUCTION

A number of homogeneous enzyme immunoassays (1,2) have been developed, some (3,4) of which have already been used for determination of antigens in clinical laboratories. However, most of them are limited to the measurement of low molecular substances such as drugs or steroid hormones.

We describe here a unique homogeneous enzyme im- munoassay for macromolecular antigens such as IgG and IgM. This method is based upon a competitive enzyme inhibition with antigen using hybrid antibody. The principle of this method is shown schematically in the following re- action.

h

-+ Hybrid Ab

Enzyme

0 I G

4

tigen exists, the complex of hybrid antibody with antigen prevents binding of the hybrid antibody to the enzyme. Therefore, the activity of the enzyme is proportional to the concentration of antigen being assayed.

MATERIALS AND METHODS

Enzyme and Chemicals

Glucose-6-phosphate dehydrogenase (NADP type) (EC 1.1.1.49) and NADP were purchased from Oriental Yeast (Tokyo, Japan). Glucose 6-phosphate and N-hydroxysuc- cinimide ester of N-(4-carboxycyclohexylmethyl)maleimide (CHMS) were purchased from Sigma Chemical Company, St. Louis, MO. Other chemicals used were of analytical grade.

inactive

Antisera

Goat antiserum to human IgM was obtained from Fuji- rebio’s Obihiro laboratory and purified to specific IgG to human IgM by affinity chromatography.

The hybrid antibody is able to bind to antigen or enzyme competitively and inhibit enzyme activity. When excess an-

@ 1987 Alan R. Liss, Inc.

Anti-GGPDH Mouse Monoclonal Antibody

BALB/c mice were immunized weekly with an intraper- itoneal injection of G6PDH (NADPH type) in complete adjuvant for 4 weeks. Spleen cells were fused with P3U1 myeloma cells by 35% PEG-1000, and hybridoma cells

78 Ashihara et al

100

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W

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c .- .- c a w 50 .- 4- 0 W a -

producing antibody, which inhibited G6PDH activity, were cloned. Ascites were obtained from BALB/c mice injected with the hybridoma cells.

-

-

Preparation of Hybrid Antibody

CHMS (1 mg) in 100 pl of dimethylformamide was added to 1 mg of anti-G6PDH mouse IgG in 1 ml of 0.1 M phosphate buffer, pH 6.3. The reaction mixture was in- cubated at 30°C for 3 hr, then the mixture was gel filtrated on a 1.5 X 45 cm column of Sephadex G-25 coarse, equi- librated with 0.1 M phosphate buffer, pH 6.3. Protein fraction was collected and concentrated to 1 mg/ml of solution using PEG-20,000. S-Acetylmercaptosuccinic anhydride ( 1 mg) in 100 p1 of dimethylformamide was added to 1 mg of anti- human IgM goat IgG in 1 ml of 0.1 M phosphate buffer. The mixture was incubated at 37°C for 2 hr. Then 100 p1 of 0.1 M hydroxylamine solution, pH 7.0, was added to the mixture. After incubation at 37°C for 1 hr, the mixture was applied onto a Sephadex G-25 column (1.5 x 45 cm) equi- librated with 0.1 M phosphate buffer, pH 6.3. The protein fractions were collected and mixed with the above-mentioned CHMS-induced anti-G6PDH mouse IgG. The mixture was incubated at 30°C for 2 hr and at 4°C overnight. After that, it was applied onto a 2.0 x 100 cm column of Sephacryl S-300, equilibrated with 20 mM phosphate-buffered saline (PBS), pH 7.0. The fractions containing the hybrid antibody in a molar ratio of 1 : 1 (anti-G6PD 1gG:anti-IgM IgG) were collected.

Assay Procedure

Hybrid antibody solution (50 pl) (80 pg/ml) was added to 50 p1 of standard solutions containing various concentrations of human IgM. After 20 min at 37"C, 1.0 ml of substrate solution, pH 8.5, containing 0.5 mM glucose 6-phosphate, 1.3 mM NADP, 0.1 M glycylglycine, and 20 mM MgC12, was added to the mixture.

RESULTS AND DISCUSSION

Anti-G6PDH mouse IgG was purified from mouse ascites in which 5 mgiml of IgG was obtained. As shown in Fig. 1, anti-G6PDH IgG inhibited 80% of the original activity of G6PDH (line A). Pepsin digestion or conjugation of anti- human IgM goat IgG to mouse IgG did not alter the binding ability of the mouse IgG to inhibit the enzyme (line B, C). However, 10 times as much concentration of hybrid antibody was required to inhibit the same amount of enzyme. This result demonstrates that conjugation decreases the binding affinity by steric hindrance. Accordingly, in subsequent assay, 4.0 pg of hybrid antibody was used.

We examined the effect of incubation time on the inhibiting activity of antibodies to G6PDH. As shown in Fig. 2, intact IgG, Fab', and the hybrid antibody inhibited the enzyme activity within 5 min.

Maximum inhibition was reached within 10 min and each antibody showed the same inhibition pattern. Enzymatic digestion or chemical modification does not alter the binding ability to the enzyme. Accordingly, the immunological re- action for a standard assay was performed for 20 min.

Hybrid antibody was prepared by the SH-maleimide method ( 5 ) which is suited for specific conjugation under mild conditions. But the hybrid antibody in this study ranged from an MW of 320,000 to over 500,000. Therefore, we also attempted to prepare a Fab-Fab conjugate using Fab- SH obtained by enzymatic digestion. However, anti-G6PDH mouse IgG was not digested to F(ab')z, but to Fab' which had no reactive SH group.

Figure 3 shows the standard curve for determination of human IgM. The measurable range for IgM was from 78 pg/ml to 1 mg/ml. Human IgG was not affected by this assay.

The present assay measured human IgM in the serum. When human serum was titrated with fixed levels of hybrid antibody and enzyme, the enzyme activity was increased in

0 10 20 30 40

Incubation Time [ min 1

Fig. 2. Effect of incubation time on the activity of the enzyme by anti- G6PDH antibodies. Activity at 25°C was measured after the enzyme was incubated at 37°C for varying time with unmodified IgG (O), Fab' (O), or hybrid antibody (A).

Homogeneous Enzyme lmmunoassay 79

the dilution of (1/2)5 to (1/2)3 (Fig. 4). In this study, we were unable to obtain a sensitive assay. However, the sen- sitivity in this method depends on the preparation of the hybrid antibody, which is difficult to prepare by chemical conjugation. Recently, J. Martinis et al. (6) made hybrid monoclonal antibodies which were able to bind to both the HB antigen and prostatic phosphatase. Our method is also able to apply this preparation; however, the hybrid monoclonal antibodies themselves are not suited to our method because it binds simultaneously both antigen and enzyme due to the flexible hinge region. Therefore, applying hybrid monoclonal antibody to our method will require chemical or immuno- chemical treatment to decrease the flexibility.

0.015

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78 312 1250 Human IgM Concentratlon [ uglml)

REFERENCES

0.015 c z 0 d m 0.010 - e

C - E 2 0.005- a

8 6 4 2 0

Serum Dilution/ [ 1/2)”

Fig. 4. were added to hybrid antibody, followed by 50 pI of enzyme solution.

Dilution of normal human serum with PBS. Fifty-microliter aliquots

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3 . Li TM, Benovic JL, Buckler RT, Burd JF: Homogeneous substrate- labeled fluorescent imrnunoassay for theophylline in serum. Clin Chem 27:22, 1981.

4. Gushaw JB, Miller JG: Homogeneous enzyme immunoassay for meth- oxalate in serum. Clin Chem 24:1032, 1978.

5 . Yoshitake S, Yamada Y, lshikawa E, Masseyeff R: Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N- hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide. Eur J Biochem 101:395, 1979.

6 . Martinis J, David GS, Wang R, Bartholomew RM: Novel applications of monoclonal antibodies. Basic Life Sci 25:129, 1983.